Characterization and Regulation of High Risk and Malignant Breast Epithelium.Breast cancer commonly develops in the epithelial cells lining the breast milk ducts. An understanding of the characteristics of high risk epithelium is critical for defining the carcinogenic pathway, for risk assessment, and for selecting and monitoring for chemoprevention therapy. To provide a more comprehensive evaluation of the high risk duct and ductal epithelium, two clinical trials are being conducted: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and cDNA Gene Expression Profile (DN Danforth, PI). This is a collaborative study in which the normal contralateral breast is being studied in women with ipselatersal breast cancer. The contralateral breast is being studied by breast duct lavage and breast duct endoscopy, including targeted endoscopic sampling of the epithelium, and the ductal epithelial cells are characterized cytologically, by gene expression profiling and protein lysate array, and for chromosomal abnormalitites by comparative genomic hybridization. The protocol is active, and eighteen subjects have been enrolled, and 41 ducts have been studied. The subjects were predominantly postmenopausal, with infiltrating ductal carcinoma (77.8 %) and were previously treated with chemotherapy (72.2 %) and tamoxifen (44.4 %). Fifty-six percent of all ducts studied did not produce nipple aspirate fluid, but were easily accessed for lavage and endoscopy. Adequate cellular material for diagnosis was obtained by ductal lavage for 16 patients (88.9 %). Cellular atypia was present in 6 ducts from 5 subjects, and 5/6 had intraductal lesions on endoscopy. Endoscopy was performed on 29 ducts (17 patients). Nine ducts (9 pts) were normal and 20 ducts (13 pts) contained intraductal lesions, most commonly intraductal adhesions or fibrous bridging. Endoscopic targeted intraductal sampling provided multiple samples of high cellularity with a relatively pure ductal epithelial content (> 91% purity) in 5/6 subjects. Followup repeat ductal lavage and ductal endoscopy at 6 - 14 months in four subjects with atypia showed essentially complete resolution of the intraductal lesions and the atypia. These findings indicate that breast duct lavage and breast duct endoscopy in the high risk breast allows a more comprehensive assessment of the ducts and ductal epithelium. The addition of targeted intraductal sampling via endoscopy may enhance acquisition of cellular material for cytologic and molecular studies. The presence of cellular atypia in the contralateral breast was dynamic, and suggests reconsideration of cytologic changes as endpoints in chemoprevention trials aimed at the high risk population. Samples for proteomic and gene expression profile studies from all specimens were frozen and will be analyzed after completion of accrual. This study was awarded an NCI Intramural Bench-to-Bedside Award to support the conduct of the trial.The second ongoing clinical trial is Protocol 02-C-00-144. Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer (DN Danforth, PI). This is a collaborative study which will promote identification and understanding of phenotypic, metabolic, and molecular changes which occur in high risk or premalignant breast epithelial cells. In this study a repository has been created of high risk breast epithelial tissues and cell lines from all major sites of breast tissue at increased risk for breast cancer. These will include: normal tissue adjacent to a breast cancer, normal tissue in the same breast at a site distant from the breast cancer, from the contralateral normal breast, from women with a strong family history of breast cancer (including BRCA1 and BRCA2 mutation carriers), from women without a history of breast cancer but with a Gail model risk estimate of breast cancer of > 1.67%, or from women with prior definitive mediastinal irradiation for lymphoma. Normal tissue will also be acquired from normal women not at increased risk for breast cancer. Short-term epithelial and fibroblast cell lines will be developed from this tissue. These cell lines and respective tissues will be placed in an NIH intramural repository of high risk breast cell lines and breast tissues which can be used to further define the carcinogenic pathway for breast cancer, including the cellular, metabolic, and molecular characteristics, for cDNA and tissue arrays, and to develop a drug discovery program for chemoprevention agents using a cell line screen of high risk breast epithelial cells. It is anticipated this will become a major resource for the study of premalignant changes in breast cancer. Importantly, these high risk cell lines and tissues represent excellent material for preclinical studies. These can be used to identify cellular characteristics which can then be examined in the high risk ductal epithelial cells from the contralateral breast model above, as well as to suggest endpoints and biomarkers for short-term chemoprevention trials. This study is being done in collaboration with Suburban Hospital, Bethesda MD, which will promote acquisition of high risk material.The mechanism of action of chemoprevention and antiproliferative agents against breast cancer are being actively studied, including the antiestrogens tamoxifen and aromatase inhibitors, the retinoids, g-interferon, and Fas antigen. Tamoxifen and the retinoids each have strong antiproliferative effects on breast cancer cells, and in combination these agents act synergistically to further inhibit growth, representing an important means for enhancing their antiproliferative effects. This has resulted in increased efficacy from both a chemopreventive and therapeutic standpoint. The effect of 4-hydroxytamoxifen (TAM) and all-trans retinoic acid (AT) in combination on proliferation of MCF-7 breast cancer cells has been studied in vitro, and it was found that TAM and AT, in combination, acted synergistically to cause a time-dependent and dose-dependent inhibition of MCF-7 cell growth. In a temporally related manner, TAM + AT acted synergistically to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis. TAM and AT each blocked cell cycle progression throughout 7 days of treatment, but without any synergistic or additive effect on this process, indicating a selective synergism for apoptosis. The negative growth factor transforming growth factor beta (TGF) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM + AT on apoptosis. TAM + AT acted synergistically to induce a 5 fold increase in TGF1 secretion over 72 hours. TGF1 alone had no apoptotic effects on these cells, but TGF1 in combination with AT acted synergistically to inhibit growth, to downregulate bcl-2 mRNA and protein expression, and to stimulate apoptosis of these cells in a manner comparable to that noted for TAM + AT. The synergism of both TAM + AT and TGF1 + AT for apoptosis was suppressed by estradiol. Coincubation of TAM + AT with anti-TGF-beta antibody, however, did not block downregulation of Bcl-2 protein expression or stimulation of apoptosis. Together these findings indicate that the synergism of AT + TAM occurs independently of TGF-beta, and that TGF-beta may interact with AT in a novel manner to provide another important antiproliferative mechanism for breast cancer cells. Because AT is also a naturally occurring hormone, these findings may have broad implications for regulation of breast cancer cell growth.The aromatase inhibitors are important antiestrogenic agents which block intracellular synthesis of estrogen, in contrast to tamoxifen which blocks estrogen-estrogen receptor interaction.